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\begin{document}

\title{Respiratory Syncytial Virus incidence and typing in the last 6 seasons
in the north of Spain (Asturias). Genetic characterization during the
SARS-Cov-2 pandemic}



\author[1]{Santiago melon}%
\author[1]{Susana Rojo}%
\author[1]{Zulema Pérez-Martínez}%
\author[1]{José María González Alba}%
\author[1]{Jose Boga}%
\author[1]{Cristina Ochoa-Varela}%
\author[2]{Maria Agustina Alonso Álvarez}%
\author[2]{Carmen Pérez Fonseca}%
\author[2]{Marta María García Clemente}%
\author[3]{José Gutierrez Rodriguez}%
\author[2]{Estibaliz Garrido García}%
\author[1]{Mercedes Rodríguez-Pérez}%
\author[1]{Marta Alvarez-Arguelles}%
\affil[1]{Hospital Universitario Central de Asturias Laboratorio de Medicina}%
\affil[2]{Hospital Universitario Central de Asturias}%
\affil[3]{Instituto de Investigacion Sanitaria del Principado de Asturias}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
Respiratory syncytial virus is associated with lower respiratory tract
infections. As several types and genotypes can circulate at the same
time, genomic characterisation is important for timely epidemiological
control and treatment measures. In the last 6 seasons (2017-2023),
191236 nasopharyngeal swabs were processed for respiratory viruses. The
incidence of RSV reached 7\% in the pre-pandemic season. RSV was most
frequent in children under 5 years of age (12.6\%), but was also
significant in those over 70 years of age (5.63\%). The measures taken
to control SARS-Cov2 infection were useful for RSV control and the
incidence decreased to 1.8\%, but caused a change in the types.
Pre-pandemic, the majority circulating types were RSV-B/RSV-B/RSV-A and
in pandemic it was RSV-B/RSV-B. In the last season, RSV-B and RSV-A were
detected in the same proportion. Genetic characterization showed three
new clades. This has been taken into account in order to take the
correct measures.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Respiratory Syncytial Virus (RSV) is a common viral pathogen that causes
64 million acute respiratory infections annually\textsuperscript{1}. RSV
is considered as a major cause of lower respiratory tract infections
amongst young children, elderly and immunocompromised adults
worldwide\textsuperscript{2-5}, and is a frequent cause of
hospitalization\textsuperscript{6,7}. In a recent systematic analysis,
it was associated to 6.6 million of acute lower respiratory infection,
where more than 1 million were hospitalized and 10000 dead in the
hospital\textsuperscript{8}. For example, in the United States, RSV
infections cause between 58,000 and 80,000 hospitalizations each year in
children under 5 years of age and between 60,000 and 160,000 in adults
over 65 years of age\textsuperscript{9}.

Based on the differences in the nucleotide sequence in the G protein,
RSV is divided into 45 genotypes. RSV-A has 15 genotypes whereas RSV-B
has 30 genotypes. Different RSV genotypes exhibit different
pathogenicities. In the same period and area, different RSV genotypes
can be co-epidemic, but most epidemics are dominated by one of the
subgroups or genotypes\textsuperscript{10}. In the context of COVID-19
pandemic with resulting quarantine and barrier measures, a drastic
reduction of RSV infections has been observed in Europe and North
America\textsuperscript{1}. Genetic characterization of RSV by direct
sequencing of sub-genomic regions or full genomic sequencing will be an
important part of RSV surveillance to monitor potential antigenic
changes in the circulating viruses that might affect the efficacy of
future immunization strategies. In general, the preferred RSV
surveillance will be an active sentinel system, with both primary care
and hospital patients being systematically sampled and tested for
RSV\textsuperscript{11}. At present, data concerning the molecular
epidemiological characteristics of RSV subtypes are
limited\textsuperscript{12}.

No effective therapy or vaccine is available to date, but vaccine
candidates and monoclonal antibodies are in late clinical
development\textsuperscript{5}. Lately, a new monoclonal antibody
(nirsevimab) has been approved\textsuperscript{13,14}. The effectiveness
of these therapies may vary depending on the type of virus. Therefore,
the reporting of RSV cases and the etiological diagnosis of the genotype
are very important for planning future public health measures and allow
appropriate treatment of the patient and avoidance of unnecessary
therapies (such as antibiotics)\textsuperscript{15}.

Here, we conducted a study detecting and genotyping RSV, circulating in
clinical samples in Asturias from 2017 to 2022. The aim of this study is
to know the incidence and distribution of RSV types and to enrich the
data of epidemiological molecular studies on RSV in Spain.

\textbf{Materials and Methods}

Samples and patients

Between September 2017 to January 2023, 191236 nasopharyngeal swabs were
collected from the same number of patients to determine the etiology of
acute respiratory infection, 50026 before SARS-CoV-2 pandemic (2017-19)
and 141216 after (2020-22).

These samples correspond to 11301 children under one year of age, 36811
children aged 1 to 5 years, 17270 children aged 6 to 15 years, 80299
adults aged 16 to 70 years and 44918 adults aged 70 years and over.

\emph{\textbf{Laboratory diagnosis}}

The samples were divided into two aliquots according to laboratory
protocols. The first (1ml) was used for conventional monolayer cell
culture (MRC-5 and mix of LLC-MK2, A549 and Hep-2), while the second
(500 \selectlanguage{greek}µ\selectlanguage{english}l) was used for viral nucleic acid detection.

\emph{Nucleic acids extraction and virus detection.}

Nucleic acids were extracted and purificated by using the automated
nucleic acid purifier Magnapure (Roche Diagnostics SL, Swizertland)
following manufacturer\selectlanguage{ngerman}´s instructions. Extracted nucleic acids were
resuspended in a final volume of 70 \selectlanguage{greek}µ\selectlanguage{english}l.

RSV genome was detected and quantificated by a multiplex real time
reverse transcription polymerase chain reaction rt-RT-PCR for
Influenzavirus A and B and RSV using type-specific primer pairs and MGB
probes (Table 1) and the TaqMan Fast 1-Step Master Mix (Life
technologies, CA). RT-PCR was performed with 5 \selectlanguage{greek}µ\selectlanguage{english}l of extracted nucleic
acids in a final volume of 10 \selectlanguage{greek}µ\selectlanguage{english}l as follows: 50\selectlanguage{ngerman}º/10', 95º/7', 45 cycles
of 95º/5'' and 60º/33''.

In addition, the human ß-globin gene was quantified in each sample in
order to evaluate sample quality and to calculate normalized viral load
in copies/10\textsuperscript{3} cells.

Table 1. Primers and probe used for detection of Influenza A (IA),
Influenza B (IB) and RSV (types A and B) and sequencing\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-11}}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Target

{\label{target}}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Primer/probe

{\label{primerprobe}}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Sequence (5' \textgreater{} 3')

{\label{sequence-5-3}}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Detection

{\label{detection}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
IA

{\label{ia}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
IA-TR-S

{\label{ia-tr-s}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
GACCRATCCTGTCACCTCTGAC

{\label{gaccratcctgtcacctctgac}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-1}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
IA-TR-A

{\label{ia-tr-a}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
AGGGCATTYTGGACAAAKCGTCTA

{\label{agggcattytggacaaakcgtcta}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
IA-FAM

{\label{ia-fam}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
TGCAGTCCTCGCTCACTGGGCACG

{\label{tgcagtcctcgctcactgggcacg}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-2}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
IB\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
IB-TR-S

{\label{ib-tr-s}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
AACATGGTAGTGAAACTGGGTGAA

{\label{aacatggtagtgaaactgggtgaa}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-3}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-4}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
IB-TR-A

{\label{ib-tr-a}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
CAGCATGCGCATTTTGGAT

{\label{cagcatgcgcattttggat}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
IB-NED

{\label{ib-ned}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
AYAACCAGATGATGGTYAAA

{\label{ayaaccagatgatggtyaaa}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-5}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
RSV-A

{\label{rsv-a}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
RSVA-TR-S

{\label{rsva-tr-s}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
GCCAGTGGCATTGCTGTAT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-6}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-7}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
RSVA-TR-A\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
CTGACTACGGCCTTGTTTGT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-8}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
RSV-B

{\label{rsv-b}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
RSVB-TR-S

{\label{rsvb-tr-s}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
GCAAGTGGTATAGCTGTAT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-9}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\par\null{\label{section-10}}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
RSVB-TR-A\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
CTGACTACAGCTTTGTTTGT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSV-A \& RSV-B\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSV-VIC\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
AGAAGTGAACAAGATCAA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Typing\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSV-A\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSVA-TR-S\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
GCCAGTGGCATTGCTGTAT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSVA-TR-A\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
CGGCCTTGTTTGTGGATAGT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSV-B\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSVB-TR-S\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
GCAAGTGGTATAGCTGTA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSVB-TR-A\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
TGACTACAGCTTTGTTTGTAGACAAC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSV-A \& RSV-B\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RSV-VIC\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
AGAAGTGAACAAGATCAA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Sequencing\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
First round\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
VSR-TIP2021-G-S\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
GCAAATGCAACCATGTCCAA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
VSR-TIP2021-G-A\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
AACTGCACTACATGTCGATTGGT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Second round\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
VSRA-G-Fwd\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
GAAGTGTTCAACTTTGTACC\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
VSRB-G-Fwd\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
AAGATGATTACCATTTTGAAGT\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
VSRA/B-G-Rev\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
CAACTCCATTGTTATTGCC\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\emph{RSV typing}

Two different RT-PCR assays, which include the VIC-labeled MGB probe and
two primers for RSV-A or RSV-B separately (Table 1), were carried out to
characterize RSV type (A and B) with the same amplification conditions
that above.

\emph{RSV characterization}

During 2021 and 2022, 116 RSV positive samples under 25 Ct were randomly
chosen and genotyped by Sanger sequencing method. A fragment of the~G
gene was amplified by a nested-PCR. The first round of amplification was
carried out using the OneStep RT-PCR kit (EURx, Poland) and specific
outer primers (Table 3). Five microliters of extracted viral genome were
added to 20 \selectlanguage{greek}µ\selectlanguage{english}l of a RT-PCR mixture according to the manufacturer's
instructions. Amplification was performed using a GeneAmp PCR system
9600 thermal cycler (Applied Byosistems, USA) with the following
conditions: 48\selectlanguage{ngerman}º/45', 95º/2', 40 cycles of 95º/30''-55º/30''-72º/30'',
and 72º/10'.

To increase sensibility, a second round of amplification was carried
out. Three microliters of the previous amplification reaction were added
to 22~\selectlanguage{greek}μ\selectlanguage{english}l of a PCR mixture containing 0.5~pmol of specific inner primers
(Table 1), 50~\selectlanguage{greek}μ\selectlanguage{english}M of each dNTPs (Gibco BRL, Carlsbad, CA, USA), and 1~U
of \emph{Taq} DNA polymerase (Gibco BRL) in PCR buffer (1x) supplied by
manufacturer. The amplification protocol was as follows: 95\selectlanguage{ngerman}º/5', 40
cycles of 95º/30''-55º/30''-72º/30'', and 72º/10'.

PCR products were analyzed by agarose gel electrophoresis, extracted by
using Montage DNA Gel Extraction Kit (Millipore, USA) and sequenced with
Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA)
supplemented with inner primers using an ABI 3130 genetic analyzer
(Applied Biosystems, USA).

Demographic data (age and sex) from those patients are in File S1.

\emph{Phylogenetic reconstructions}

Nucleotide sequences were translated and aligned using the MUSCLE
algorithm implemented in MEGA. Sequences (147 to 396 nucleotides)
generated in this work have been deposited in GenBank with the following
accession numbers:OR493270-OR493385.

For type characterization, phylogenetic trees were constructed using
ModelFinder, tree reconstruction and ultrafast bootstrap (1000
replicates) with IQ-TREE 2.1.3. RSV reference sequences used were in
File S2. The best-fit nucleotide substitution model GTR+F+G4 was
identified according to Bayesian information criterion. Bootstrap values
were estimated using the SH test and ultrafast bootstrap with 1000
replicates. To identify the phylogenetic relationship, 8373 G gene
sequences type A and 5339 type B available in the GenBank database (one
sequence of each type used for the Basic Local Alignment Search Tool
(BLAST) in March 2023) were used (File S2). Transmission clusters were
defined as viral sequences circulating in Asturias grouped into a single
and well supported monophyletic clade with \textgreater{}90\% bootstrap
values. To search for mutations, the sequences were compared with ON1
(JN257693) and BA9 (DQ227395).

\emph{Statistical analyses}

Statistical tests were performed using GraphPad InStat v.3 (GraphPad
Software, USA). These tests were considered significant when p value was
\textless{}0.05.

\textbf{Results}

Incidence and subtypes of RSV

Of the 191236 samples tested in the 6 seasons, 6203 (3.3\%) were
positive. From them, 1433 (23.1\%) were typed as RSV-A, and 3492
(56,3\%) as RSV-B. Table 2 and Figure 1 show the incidence by season and
age, and types found.

Table 2. Number and Rate of RSV detected each season by age and types
(A/B).\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Seasons}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{\textless{}1 year old}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{1-5 years}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{6-15 years}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{16-70 years}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{\textgreater{}70 years}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Total}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
S1 2017-18\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
335/1704 (19,7\%) 6A/175B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
375/3848 (9,7\%) 2A/185B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
75/1605 (4,7\%) 0A/37B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
207/3160 (6,6\%) 4A/107B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
183/1998 (9,2\%) - /91B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1175/12315 (9,5\%) 12A/595B\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
S2 2018-19\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
384/1991 (19,3\%) 11A/344B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
386/4944 (7,8\%) 8A/341B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
43/1958 (2,2\%) - /32B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
163/4040 (4,0\%) 1A/128B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
202/3083 (6,6\%) 1A/171B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1178/16016 (7,4\%) 21A/1016B\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
S3 2019-20\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
364/1664 (21,9\%) 206A/45B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
528/4600 (11,5\%) 310A/90B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
45/2395 (1,9\%) 21A/3B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
148/8642 (3,4\%) 59A/20B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
150/4414 (3,4\%) 58A/26B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1235/21695 (5,7\%) 655A/184B\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
S4 2020-21\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
65/1616 (4,0\%) 3A/60B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
269/7347 (3,7\%) 12A/240B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3/2906 (0,1\%) 2A/1B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
12/3240 (0,4\%) 0A /11B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
9/1497 (0,6\%) -/9B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
358/16606 (2,2\%) 17A/321B\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
S5 2021-22\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
200/2901 (6,9\%) 21A/170B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
610/10770 (5,7\%) 29A/538B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
26/5549 (0,5\%) 1A/20B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
43/53568 (0,1\%) 5A/33B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
21/28372 (0,1\%) -/20B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
900/101160 (0,9\%) 56A/781B\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
S6 2022-J23\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
327/1425 (22,9\%) 157A/156B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
638/5302 (12,0\%) 343A/263B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
93/2857 (3,3\%) 52A/32B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
117/7649 (1,5\%) 41A/60B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
183/5554 (3,3\%) 79A/84B\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1358/22787 (6,0\%) 672A/595B\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Total\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1675/11301 (14,8\%) 404A/950B\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2805/36811 (7,6\%) 704A/1657B\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
285/17270 (1,7\%) 76A/125B\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
690/80299 (0,9\%) 110A/359B\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
748/44918 (1,7\%) 138A/401B\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6203/190579 (3,3\%) 1433A/3492B\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

S: Season; J: January

In the three pre-pandemic SARS-Cov-2 seasons 3587 (7.1\%) of the samples
had RSV, and in the pandemic seasons 2616 (1.8\%) (p\textless{}0.0001).

On the other hand, RSV was detected in 3480 (9.1\%) children under 6
years of age and in 1438 (1.14\%) adults over 15 years of age
(p\textless{}0.0001).

Finally, type A accounted for 1.97\% in S1, 2.2\% in S2, 78.6\% in S3,
5.02\% in S4, 6.69\% in S5 and 53\% in S6 (p\textless{}0.0001).

Figure 1. Incidence of RSV by season and age (A) or types (B).

RSV genotypes

Phylogenetic analysis of 116 sequenced RSV isolates showed that 51 RSV-A
(9 from 2021 and 42 from 2022) viruses belong to the ON1 /genotype and
65 RSV-B (24 from 2021 and 41 from 2022) viruses belong to BA9 (Figure
S1).

The ON1 was divided into two major clades and four small clades. The
first major clades were related to strains from Kenia 2015, France 2021
and USA 2022 (30 patients), and the second to Netherlands 2021, Slovenia
2021 and France 2022 (15 patients). The small clades were related to
strains from Bangladesh 2012 (1 patient), China 2018 (1 patient),
Slovenia 2021(1 patients), USA 2022 (3 patients) (Figure 2A).

The BA9 was divided in one major clade related to strains from China
2019, Japan 2019, Italy 2021, USA 2021 Australia 2022, and Austria 2022
(22 patients 2021/ 41 patients 2022), and in two small clades all of
2021 related to strains from Germany 2017, Spain 2018, Thailand 2019 and
China 2020 (1 patient), China 2021(1 patient) (Figure 2B).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 2} . Phylogenetic tree of genomes of RSV-A (A) and RSV-B
(B) from Asturias (black circles) and NCBI. The geographical origins of
the NCBI sequences phylogenetically related to those of Asturias are
indicated.

In 2021, a new RVS-B sublineage spread in Asturias, characterized by the
S263G mutation). In 2022, two new sublineages from ON1 spread in
Asturias, one characterized by the S299I mutation (n=8) and the other by
the K205N and Y280H mutations (n=26) (Table 3, Figure 3).

\textbf{Table 3.} Characteristics of the sublineages identified during
2021--22 in Asturias (Spain).\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\textbf{Sublineage} & \textbf{n} & \textbf{Detection date} &
\textbf{Sex} & \textbf{Sex} & \textbf{Age (years)} & \textbf{Age
(years)} & \textbf{Age (years)} & \textbf{Age (years)} &\tabularnewline
\midrule
\endhead
& & & \textbf{Female} & \textbf{Male} & \textbf{\textless{}1} &
\textbf{1-5} & \textbf{6-15} & \textbf{16-70} &
\textbf{\textgreater{}70}\tabularnewline
ON1+K205N+Y280H & 26 & 31/10/2022 & 14 & 12 & 5 & 11 & 5 & 2 &
3\tabularnewline
ON1+S299I & 8 & 13/11/2022 & 2 & 6 & 2 & 2 & 2 & 1 & 1\tabularnewline
BA9+S263G & 13 & 06/10/2021 & 4 & 9 & 1 & 3 & & 4 & 5\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3.} A) New ON1 sublineage that spread in Asturias,
characterized by the K205N and Y280H mutations. B) New ON1 sublineage
that spread in Asturias, characterized by the S299I mutation. C) New BA9
sublineage that spread in Asturias, characterized by the S263G mutation.
All of them identified with red branches.

\textbf{Discussion}

Respiratory Syncytial virus is the most common cause of serious
respiratory infection in infants. Reinfections occur commonly, including
in older adults\textsuperscript{16}. In recent years, the importance and
severity of RSV infections in children as well as adults has been
demonstrated and appreciated\textsuperscript{1-7}. RSV-associated
in-hospital mortality increases exponentially with age, posing a greater
risk for older adults, particularly frail and high-risk
patients\textsuperscript{8,9,17,18} .Moreover, RSV infection may result
in greater morbidity and mortality among older hospitalized adults than
influenza\textsuperscript{19}. RSV can also causes substantial
outpatient illness with lower respiratory tract involvement. Belongia et
al found the virus in the 11\% of patients\textsuperscript{20}. In a
previous job in our Hospital this incidences reached 14\% when children
was principally studied\textsuperscript{21}.

It has been already reported that during the SARS-Cov2 pandemic,
mitigation measures were associated with marked reductions in
non-COVID-19 respiratory infections\textsuperscript{9,22}. In this
study, the overall incidence over 6 seasons was 3.3\%, but a large
difference was observed before and after the pandemic: in the first
three seasons, the incidence reached 7.1\% and decreased to 1.8\% during
the pandemic years

RSV was more common in children (9.1\%) than in adults (1.14\%), but can
be found in any age group. Moreover, if we analyze the first two seasons
(before the pandemic), the incidence in those over 70 years of age is
similar to that in children aged one to five years. And the incidence is
not negligible in the rest of the age groups. As noted above, the
measures taken during the pandemic reduced viral transmission,
especially in adults, where more pressure and control was exerted. The
return to normality, even with the lessons learned, suggests that the
incidence of RSV infection is likely to rise again.

RSV may be associated with escalation in respiratory support and an
increased level of support in living situation at
discharge\textsuperscript{23}. RSV disease burden in adults aged more
than 60 years in high-income countries is higher than previously
estimated\textsuperscript{24}. RSV hospitalizations were associated with
increased attributable short-term and long-term healthcare
costs\textsuperscript{1}. Therefore, interventions that could prevent
RSV may reduce healthcare burden. Understanding the clinical features
and symptomatology of RSV infection can help to address the challenges
related to case identification and management and allows for the
implementation of appropriate preventive measures. For example, in this
study incidence decreased from 5.6\% pre-pandemic to 0.6\% during
SARS-Cov2 pandemic.

Since two types and several genotypes have been described and the
behaviour of each may be different, an exact classification is
necessary. Types RSV-A and RSV-B are simultaneously present in most
outbreaks, but RSV-A is associated with severe disease. In RSV-A
dominant years typically started, reached its peak and lasted than in
RSV-B dominant years\textsuperscript{8,25}. Several distinct genotypes
within these types predominate within a community; the dominant strains
shift yearly, perhaps accounting for frequent
reinfections\textsuperscript{25,26}. Previous RSV infection does not
appear to protect against reinfection\textsuperscript{27}. On the other
hand, several substitutions in fusion protein reduce nirvesimab
susceptibility\textsuperscript{28}, the new monoclonal antibody approved
to immunize against RSV.

In this job, RSV-A and RSV-B were found simultaneously, but with very
different incidence. In the first two seasons, RSV-B was predominant,
but in 2019 (pre-pandemic SARS-Cov-2) there was a subtype shift and
RSV-A was detected in 78\% of cases, suggesting greater severity of
symptoms. The measures taken in the pandemic significantly reduced
respiratory infections, as discussed above\textsuperscript{9,22} .In the
first two years of the pandemic a new subtype shift was observed and
again RSV-B became dominant. In the last season (2022-23)
epidemiological control measures were again relaxed and both subtypes
were observed to circulate in the same proportions.

Next season we will have to watch out whether RSV-A (or certain
genotypes of this type) replaces RSV-B, as suggested by other
studies\textsuperscript{29,30}, as a worsening of symptoms is to be
expected. This is why

sequencing of viral strains is relevant not only for taxonomic purposes,
but in order to better understand the epidemiology as well as the
development of therapeutic and preventive strategies.

The genotyping analysis of RSV was used to characterize which genotypes
circulated in Asturias and to investigate whether phylogenetic clusters
occurred in the study period.

RSV genotypes ON1 (type A) and BA9 (type B) became the predominant
genotypes worldwide from 2015 and were the only genotypes detected in
the period 2021-2022 in Asturias. Variants of these genotypes are
constantly generated, so it is necessary to identify
them\textsuperscript{31}. Numerous variants of the ON1 genotype have
been reported worldwide, with various amino acid
substitutions\textsuperscript{32,33}. In a last study in USA in 2023,
Goya et al did not find specific changes\textsuperscript{34}.

In Asturias, in the ON1 genotype, the gain of the K205N mutation that
was only found in two NCBI strains (ON237320 Argentina 2017 and KX827403
Guatemala 2013) and the Y280H mutation (common in the world and found in
Spain) formed a separate sublineage in 26 sequences. This variant was
found in 2022 in a similar number of men and women, mostly being under 6
years of age. Besides, the gain of the S299I mutation in 8 sequences
found in strains without duplication of ON1 (KT765684-KT 765686,
OK458608-OK458615 Kenia 2006-2010) generated another new sublineage.
This variant was found in 2022 mostly in men, also mostly in children
less than 6 years of age, but not exclusively.

From VRS-B, in Europe the BA9 genotype is always the predominant, as was
also reported in Catalonia ( Spain)\textsuperscript{32,35}. In Asturias,
in 12 sequences the BA9 genotype, the gain of the mutation S263G, that
was only found in two NCBI strains (OK078747 Pakistan 2010 and MH606068
Croatia 2017), formed a separate sublineage. This variant was found in
2021 in women under 6 years of age; it continued to be disseminated in
2022 in people over 65 years of age mainly in men.

Therefore these new sublinages can be found in any group of individuals.
On the other hand, variants without these mutations were previously
detected in Asturias, supporting the idea that they were locally
generated, in contrast to the finding of Goya et al\textsuperscript{34}.

When the GenBank sequences were examined, no new mutations were
observed, but rare mutations appeared in new lineages, which could be
limited to local transmission. New substitutions may contribute to
antigenic escape, promote transmissibility, or be the result of the
founder effect in a vulnerable population. Therefore, performing
genotypic characterisation studies locally and sharing them with the
rest of the scientific community should be a practice to be implemented
in the control of this type of infections.

Limitations of the study are that few clinical data were available, that
the number of patients sequenced is limited, that changes in the
frequency of circulation of RSV genotypes in previous seasons cannot be
addressed and that the analysis was based only on a fragment of the G
gene.

\textbf{Conclusion}

In summary, although RSV was more frequent in children, it can infect at
any age. Both subtypes (RSV-A and RSV-B) were found with B/B/A/B/B/A-B
pattern. Genomic analyses did not detect any novel mutations, but did
detect the emergence of new lineages with rare mutations in the data
available to date. These lineages may influence the preventive
treatments that are attempted.

This study provides an overview of the genetic variation of circulating
RSV strains in Asturias with the identification of new ON1 and BA9
lineages. Ongoing and long-term molecular epidemiological studies for
the detection of circulating and emerging genotypes in combination with
clinical data are needed to gain a better understanding of the
underlying genetic and antigenic mechanisms of RSV infection.

\textbf{Acknowledgments:} Project partially supported by Grupin
IDI/2021/000033

\textbf{References}

1. Mac S, Shi S, Millson B, Tehrani A, Eberg M, Myageri V, et~al. Burden
of illness associated with Respiratory Syncytial Virus (RSV)-related
hospitalizations among adults in Ontario, Canada: A retrospective
population-based study. Vaccine. 7 de agosto de 2023;41(35):5141-9.

2. Fretzayas A, Moustaki M. Etiology and clinical features of viral
bronchiolitis in infancy. World J Pediatr. agosto de 2017;13(4):293-9.

3. van Houten CB, Oved K, Eden E, Cohen A, Engelhard D, Boers S, et~al.
Observational multi-centre, prospective study to characterize novel
pathogen-and host-related factors in hospitalized patients with lower
respiratory tract infections and/or sepsis - the \selectlanguage{ngerman}«TAILORED-Treatment»
study. BMC Infect Dis. 7 de agosto de 2018;18(1):377.

4. Nam HH, Ison MG. Respiratory syncytial virus infection in adults.
BMJ. 10 de septiembre de 2019;366:l5021.

5. Coppée R, Chenane HR, Bridier-Nahmias A, Tcherakian C, Catherinot E,
Collin G, Lebourgeois S, Visseaux B, Descamps D, Vasse M, Farfour E.
Temporal dynamics of RSV shedding and genetic diversity in adults during
the COVID-19 pandemic in a French hospital, early 2021. Virus Res. 2022
Sep 29;323:198950. doi: 10.1016/j.virusres.2022.198950. Epub ahead of
print. PMID: 36181977; PMCID: PMC9519364.

6. Ghazaly M, Nadel S. Characteristics of children admitted to intensive
care with acute bronchiolitis. Eur J Pediatr. junio de
2018;177(6):913-20.

7. Meissner HC. More on Viral Bronchiolitis in Children. N Engl J Med.
22 de septiembre de 2016;375(12):1200.

8. Li T, Fang H, Liu X, Deng Y, Zang N, Xie J, et~al. Defining RSV
epidemic season in southwest China and assessing the relationship
between birth month and RSV infection: A 10-year retrospective study
from June 2009 to May 2019. J Med Virol. julio de 2023;95(7):e28928.

9. Hamid S, Winn A, Parikh R, Jones JM, McMorrow M, Prill MM, et~al.
Seasonality of Respiratory Syncytial Virus - United States, 2017-2023.
MMWR Morb Mortal Wkly Rep. 7 de abril de 2023;72(14):355-61.

10. Chen G. Lan M, Lin S, Zhang Y, Zhang D, Weng Y, Zheng K. Genome
analysis of human respiratory syncytial virus in Fujian Province,
Southeast China. Infect Genet Evol. 2022 Sep;103:105329. doi:
10.1016/j.meegid.2022.105329. Epub 2022 Jul 3. PMID: 35788050.

11. Teirlinck AC. Broberg EK, Stuwitz Berg A, Campbell H, Reeves RM,
Carnahan A, Lina B, Pakarna G, Bøås H, Nohynek H, Emborg HD, Nair H,
Reiche J, Oliva JA, O'Gorman J, Paget J, Szymanski K, Danis K, Socan M,
Gijon M, Rapp M,Havlí\selectlanguage{polish}č\selectlanguage{english}kov\selectlanguage{ngerman}á M , Trebbien R, Guiomar R, Hirve SS, Buda S,
van der Werf S, Meijer A, Fischer TK. Recommendations for respiratory
syncytial virus surveillance at the national level. Eur Respir J. 2021
Oct 1;58(3):2003766. doi: 10.1183/13993003.03766-2020. PMID: 33888523;
PMCID: PMC8485062.

12. Staadegaard L. Caini S, Wangchuk S, Thapa B, de Almeida WAF, de
Carvalho FC, Njouom R, Fasce RA, Bustos P, Kyncl J, Novakova L, Caicedo
AB, de Mora Coloma DJ, Meijer A, Hooiveld M, Huang S, Wood T, Guiomar R,
Rodrigues AP, Danilenko D, Stolyarov K, Lee VJM, Ang LW, Cohen C, Moyes
J, Larrauri A, Delgado-Sanz C, Le MQ, Hoang PVM, Demont C, Bangert M,
van Summeren J, Dückers M, Paget J. The Global Epidemiology of RSV in
Community and Hospitalized Care: Findings From 15 Countries. Open Forum
Infect Dis. 2021 Mar 30;8(7):ofab159. doi: 10.1093/ofid/ofab159. PMID:
34337092; PMCID: PMC8320297.

13. Strine MS, Wilen CB. Game over for RSV? Sci Immunol. 8 de junio de
2023;8(84):eadi8764.

14. Simões EAF, Madhi SA, Muller WJ, Atanasova V, Bosheva M, Cabañas F,
et~al. Efficacy of nirsevimab against respiratory syncytial virus lower
respiratory tract infections in preterm and term infants, and
pharmacokinetic extrapolation to infants with congenital heart disease
and chronic lung disease: a pooled analysis of randomised controlled
trials. Lancet Child Adolesc Health. marzo de 2023;7(3):180-9.

15. Menasalvas Ruiz AI. Respiratory syncytial virus infection in
COVID-19 times: Trick or threat? Enferm Infecc Microbiol Clin (Engl Ed).
2023;41(6):319-20.

16. Falsey AR, Walsh EE. Respiratory syncytial virus Prefusion F
vaccine. Cell. 20 de julio de 2023;186(15):3137-3137.e1.

17. Nguyen-Van-Tam JS, O'Leary M, Martin ET, Heijnen E, Callendret B,
Fleischhackl R, et~al. Burden of respiratory syncytial virus infection
in older and high-risk adults: a systematic review and meta-analysis of
the evidence from developed countries. Eur Respir Rev. 31 de diciembre
de 2022;31(166):220105.

18. Heppe-Montero M, Gil-Prieto R, Del Diego Salas J, Hernández-Barrera
V, Gil-de-Miguel Á. Impact of Respiratory Syncytial Virus and Influenza
Virus Infection in the Adult Population in Spain between 2012 and 2020.
Int J Environ Res Public Health. 9 de noviembre de 2022;19(22):14680.

19. Ackerson B, Tseng HF, Sy LS, Solano Z, Slezak J, Luo Y, et~al.
Severe Morbidity and Mortality Associated With Respiratory Syncytial
Virus Versus Influenza Infection in Hospitalized Older Adults. Clin
Infect Dis. 2 de julio de 2019;69(2):197-203.

20. Belongia EA, King JP, Kieke BA, Pluta J, Al-Hilli A, Meece JK,
et~al. Clinical Features, Severity, and Incidence of RSV Illness During
12 Consecutive Seasons in a Community Cohort of Adults \selectlanguage{english}[?]60 Years Old.
Open Forum Infect Dis. diciembre de 2018;5(12):ofy316.

21. Gomez-Novo M, Boga JA, Alvarez-Arguelles ME, Rojo-Alba S, Fernandez
A, Menendez MJ, et~al. Human respiratory syncytial virus load normalized
by cell quantification as predictor of acute respiratory tract
infection. J Med Virol. mayo de 2018;90(5):861-6.

22. Agha R, Avner JR. Delayed Seasonal RSV Surge Observed During the
COVID-19 Pandemic. Pediatrics. septiembre de 2021;148(3):e2021052089.

23. Hill-Ricciuti A, Walsh EE, Greendyke WG, Choi Y, Barrett A, Alba L,
et~al. Clinical impact of healthcare-associated respiratory syncytial
virus in hospitalized adults. Infect Control Hosp Epidemiol. marzo de
2023;44(3):433-9.

24. Savic M, Penders Y, Shi T, Branche A, Pircon JY. Respiratory
syncytial virus disease burden in adults aged 60\selectlanguage{english} years and older in
high-income countries: A systematic literature review and meta-analysis.
Influenza Other Respir Viruses. enero de 2023;17(1):e13031.

25. Gilca R, De Serres G, Tremblay M, Vachon ML, Leblanc E, Bergeron MG,
et~al. Distribution and clinical impact of human respiratory syncytial
virus genotypes in hospitalized children over 2 winter seasons. J Infect
Dis. 1 de enero de 2006;193(1):54-8.

26. Robertson M. Eden JS, Levy A, Carter I, Tulloch RL, Cutmore EJ,
Horsburgh BA, Sikazwe CT, Dwyer DE, Smith DW, Kok J. The
spatial-temporal dynamics of respiratory syncytial virus infections
across the east-west coasts of Australia during 2016-17. Virus Evol.
2021 Jul 22;7(2):veab068. doi: 10.1093/ve/veab068. PMID: 34532066;
PMCID: PMC8438877.

27. Hall CB, Walsh EE, Long CE, Schnabel KC. Immunity to and frequency
of reinfection with respiratory syncytial virus. J Infect Dis. abril de
1991;163(4):693-8.

28. Ahani B, Tuffy KM, Aksyuk AA, Wilkins D, Abram ME, Dagan R, et~al.
Molecular and phenotypic characteristics of RSV infections in infants
during two nirsevimab randomized clinical trials. Nat Commun. 19 de
julio de 2023;14(1):4347.

29. Otieno JR. Kamau EM, Agoti CN, Lewa C, Otieno G, Bett A, et al.
Spread and evolution of respiratory syncytial virus A genotype ON1,
coastal Kenya, 2010-2015. Emerg Infect Dis. 2017;23:264--71.

30. Viegas M. Goya S, Mistchenko AS. Sixteen years of evolution of human
respiratory syncytial virus subgroup A in Buenos Aires, Argentina: GA2
the prevalent genotype through the years. Infect Genet Evol.
2016;43:213--21.

31. Luo Q. Li M, Li A, Gong C, Dong M, Huang Q, Luo M, Zhang H, Huang F.
Genetic diversity and epidemiological features of respiratory syncytial
virus, Beijing, 2015-2019: A multicenter and all-age groups study. J
Infect. 2022 Jul;85(1):75-85. doi: 10.1016/j.jinf.2022.04.046. Epub 2022
May 6. PMID: 35533834.

32. Lin TY. Chi H, Kuo CY, Tsai HP, Wang JR, Liu CC, Shen CF. Outbreak
of respiratory syncytial virus subtype ON1 among children during
COVID-19 pandemic in Southern Taiwan. J Microbiol Immunol Infect. 2022
Dec;55(6 Pt 2):1168-1179. doi: 10.1016/j.jmii.2022.08.015. Epub 2022 Sep
13. PMID: 36137926.

33. Gaymard A,. Bouscambert-Duchamp M, Pichon M, Frobert E, Vallee J,
Lina B, Casalegno JS, Morfin F. Genetic characterization of respiratory
syncytial virus highlights a new BA genotype and emergence of the ON1
genotype in Lyon, France, between 2010 and 2014. J Clin Virol. 2018 May;
102:12-18. doi: 10.1016/j.jcv.2018.02.004. Epub 2018 Feb 6. PMID:
29471266.

34. Goya S, Sereewit J, Pfalmer D, Nguyen TV, Bakhash SAKM, Sobolik EB,
et~al. Genomic Characterization of Respiratory Syncytial Virus during
2022--23 Outbreak, Washington, USA - Volume 29, Number 4---April 2023 -
Emerging Infectious Diseases journal - CDC. {[}citado 30 de agosto de
2023{]}; Disponible en:
https://wwwnc.cdc.gov/eid/article/29/4/22-1834\_article

35. Gimferrer L. Campins M, Codina MG, Martin Mdel C, Fuentes F,
Esperalba J, Bruguera A, Vilca LM, Armadans L, Pumarola T, Anton A.
Molecular epidemiology and molecular characterization of respiratory
syncytial viruses at a tertiary care university hospital in Catalonia
(Spain) during the 2013-2014 season. J Clin Virol. 2015 May;66:27-32.
doi: 10.1016/j.jcv.2015.02.018. Epub 2015 Feb 26. PMID: 25866332.

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